Our goal is to understand the molecular mechanism of the photochemical events in visual excitation. We want to understand how rhodopsin shifts the absorption of its 11-cis retinal protonated Schiff base chromophore from 440 nm in solution to 500 nm in rod pigments, and how this absorption maximum is regulated in blue- and red-absorbing pigments. We want to elucidate the mechanism of the 11-cis to all-trans isomerization and of energy storage in the primary photoproduct. We also want to know how this stored energy is used to drive the subsequent kinetic decay processes and protein conformational changes that activate transducin. These goals will be addressed by performing time-resolved and low-temperature resonance Raman experiments to study the structure and kinetics of the chromophore in rhodopsins and through femtosecond (10-15 s) transient absorption spectroscopy. The specific aims are: (1) Resonance Raman spectra will be obtained of rhodopsin site specific mutants at 77 K. By determining which mutations alter the vibrational structure of the chromophore in rhodopsin, we will identify the important residues and interactions that produce the opsin shift. By examining the effect of these mutations on bathorhodopsin, we will identify the residues that are responsible for energy storage. (2) Resonance Raman microscope spectra will be obtained of blue and red absorbing pigments from individual photoreceptor cells at 77 K. These data will enable us to examine the molecular mechanism of the opsin shift in these natural pigments. (3) Femtosecond dynamic absorption spectroscopy with 20 fs time-resolution will be used to determine the time scale for the first step in vision. (4) Picosecond time-resolved resonance Raman spectroscopy will be used to study the structure of the primary photoproducts in vision at room temperature to understand the mechanism of energy storage and to understand the mechanism of the isomerization process. (5) Resonance Raman spectra will be obtained of rhodopsins regenerated with dicis-retinals to probe the ability of the chromophore binding site to accommodate different chromophore geometries and Schiff base orientations. This will help to define the steric and electrostatic/hydrogen-bonding requirements for the formation of a pigment. 6) Finally, time-resolved Raman experiments will be performed on the Microsecond to millisecond time scale to obtain high-quality spectra of the lumi, meta I and meta II intermediates. The structure of the retinal chromophore in each intermediate will be determined and the decay kinetics between these species will be analyzed to determine the kinetic and thermodynamic properties of these transitions.